Study of behavioral patterns and infection analyses in anopheline species involved in the transmission of malaria in Buriticupu and São José de Ribamar municipality, Maranhão State, Brazil

Anopheles darlingi Root and Anopheles aquasalis Curry are the main vectors of malaria that occur in the State of Maranhão. Entomological surveys based on the behavior and infectivity of these vectors are important for the elaboration of disease control strategies. The objectives of this work were to study the behavioral patterns of mosquitoes, determining population and hematophagic peaks, dietary preferences, infectivity rate and characterization of breeding sites in two municipalities in the State of Maranhão: Buriticupu and São José of Ribamar. Larvae and pupae were collected in breeding sites and adult females in home environments. Mosquito behavior, their dietary preferences and Plasmodium spp. infection rates were analyzed. The vegetation and physicochemical patterns in the breeding sites found are in agreement with those described for species from the Amazon region and the Brazilian Atlantic coast. Anopheles darlingi was the most prevalent mosquito in Buriticupu breeding and home environments. This species was found mainly fed on human blood and naturally infected with Plasmodium vivax Grassi & Feletti and Plasmodium falciparum Welch. Anopheles aquasalis was more frequent in breeding sites in São José de Ribamar, as well as in home environments, whose specimens were mainly fed with human and bird blood. The main peaks of mosquito occurrence in Buriticupu were between 6 pm to 9 pm and in São José de Ribamar we did not record a definite peak. In the first municipality A. darlingi showed dominance over Anopheles albitarsis Lynch Arribálzaga s.l., Anopheles oswaldoi Peryassú, Anopheles nuneztovari Gabaldón and Anopheles evansae Brèthes, besides presenting a correlation with rainfall. In the second municipality, A. aquasalis was dominant over A. albitarsis s.l. and there was a correlation between these two species and the rainy season. We conclude that the collected data contribute to elucidate the dynamics of malaria transmission in the region and guide the control actions directed to the elimination of the disease in the country.

The studies of malaria vectors are relevant for the biology knowledge, the behavior of species and the dynamic transmission of plasmodia parasites in each locality, helping in this way to elucidate the disease chain, the prediction of outbreaks and the guidance of control actions (BarBosa et al. 2014(BarBosa et al. , 2016. The entomological parameters such as density, anthropophilia, zoophilia, peaks of hematophagic activity, seasonal variations, infection rate and blood-feeding patterns are among the most frequent for the determination of the vector species (BarBosa et al. 2014).
In these studies, the relationship between the reduction in density of this group and the Amazonian rainy period was demonstrated, being more evident for the adult forms than the immature forms, since in the rainy season these insects are displaced from permanent breeding sites, from the mainland for lakes and ponds that form along river banks during floods. As the waters rise above the level of the flooded areas they originate the temporary breeding sites located in the middle of the forest. The availability of shelters or breeding sites in the rainy season facilitates the increase of breeding sites and the extension of anopheline occurrence, with a reflection on the prevalence of malaria, as it provides greater contact between man and vector. This is due to the fact that in regions where permanent breeding sites are distanced from human dwellings during the rainy season by the formation of temporary breeding sites, these are close to human dwellings intensifying human contact / vector. The pioneering studies performed by Galvão et al. (1942) and Deane et al. (1948) concluded that the intense reproduction of A. darlingi during the rainy season is associated with the numerous breeding sites in the region during this period, as well as the presence of this vector associated with anthropic action, which promotes changes in the natural habitat of mosquitoes.
These factors condition the emergence of new cases and the onset of epidemics. The process of marked environmental changes, according to Póvoa et al. (2003), in the natural environment of the municipality of Belém, conditioned the resurgence of A. darlingi from the 90s (twentieth century), after more than twenty years without registration of this species in this area. With environmental impacts, new outbreaks of malaria transmission emerged with possible influence on the cast of local species and changes in the distribution pattern, biology and behavior of these mosquitoes.
The disordered growth of many cities in malaria-endemic areas with inadequate housing conditions and the pressure of human populations, especially the poorest, on the environment, are conditioning factors for malaria maintenance. vittor et al. (2006, 2009) demonstrated in Peru that the pressure of human occupations and activities on the environment positively favors the A. darlingi vector that finds new conditions for proliferation, increasing the risks of disease transmission. These conditions were also recorded by taDei et al. (2017) in many Brazilian Amazonian municipalities, which are responsible for the onset of epidemic outbreaks of malaria and their expansion to the major urban centers of the region.
The State of Maranhão, for example, illustrates this scenario well, because besides being considered the poorest in Brazil (iPea 2019), it has significant areas with environmental degradation where there is active transmission of malaria, both in the coastal range of its territory and in the São José de Ribamar, which accounts for the majority of cases of the disease on the Atlantic coast and Buriticupu in the intracontinental zone. The aid of this paper was study the behavioral patterns of anopheline species in Buriticupu and São José de Ribamar municipalities, in Maranhão state, determining peaks of hematophagic activities, bloodfeeding preferences, natural infection rates, breeding sites characterization; the associations among the climate conditions and the presence of the Anopheles species; and the associations among the presence of the Anopheles species. The data of this work are part of a big project which studies the areas of malaria transmissions in Maranhão, being the first publication of the set or group performed by Barros et al. (2015), in which was made the first register for A. aquasalis in Buriticupu. Thus, the data that mention the presence of this vector in that municipality was created from the set of this project.

Study areas
The studies were conducted monthly from January in 2006 to February in 2011, in two localities of Maranhão State in Brazil: A. Buritizinho village (4º30'34,1" S; 46º49'27,3" W) in municipality of Buriticupu. This locality is intercontinental; 300 km far away from the north Atlantic coast of Brazil and it is located between the borders of Pindaré and Buriticupu rivers, therefore, it is a semi-urban locality The access that place is by BR-222 Highway and by EF-315 dos Carajás Railroad.
B. Guarapiranga village (02º40'52,1" S; 44º08'43,9" W) it is a semi-urban locality in municipality of São José do Ribamar. It is located in the coastal area of the Atlantic Ocean, inside of the Arraial Bay, in São Luís island complex. This area suffers the influence of tidal pulse and of several rivers whose outflow in the bay itself.

Mosquito sampling
Immature mosquitoes. The breeding sites selected for the study were located within 500 meters of human dwellings. In Buriticupu, these breeding sites were mainly characterized as riverbanks and backwaters. In São José de Ribamar, the most common breeding sites were marshes and mangroves.
The immature stages of anopheline were collected in their breeding sites between 7:00 am and 9:00 am through active search using a mosquito larvae dipper. With the obtained data we calculate the Larvae Index per Man Hour -LIMH by the formula: Nevertheless, the extra domicile corresponds to an area greater than 200 meters radius. The techniques used for catching adult females were by Castro's aspirator: I) human landing catches (HLC), by trained technicians using personal protective equipment, and; II) active search for resting mosquitoes inside houses and in locations where there were animals taking a rest. Both were conducted during a period of time from 6:00 pm to 6:00 am (12 h), stratified or separated by a timetable or schedule; and a time from 6:00 pm to 10:00 pm (4 h). The studies were conducted in six human dwellings in each locality. There were obtained temperature data, relative humidity in the gathering environments, by means of digital thermo-hygrometers, and besides rainfall, in the database of the Instituto Nacional de Meteorologia -INMET.
The collection of mosquito samples conducted in this study is part of the routine work of technicians of the Fundação Nacional de Saúde -FUNASA, authorized by the Instituto Chico Mendes de Conservação da Biodiversidade -ICMBIO (SISBIO Number 18281-1).

Mosquito identification
The collected mosquitoes were taken to the entomology laboratory of the Universidade Federal do Maranhão -UFMA, in São Luís, for identification through the dichotomous keys of Forattini (2002).

Breeding site characterization
Breeding water information such as: temperature, hydrogen potential, electric conductivity, total dissolved solids, salinity, nitrogen compounds, ammonia and phosphorus, as well as the registration of aquatic plants, were also acquired by a descriptive characterization. The identification of vegetable was conducted in laboratories of the Universidade Federal do Maranhão -UFMA, in São Luís.

Blood-feeding preferences
siqueira's (1960) precipitin technique with modifications was made in order to identify the blood-feeding. Just some female examples of A. darlingi and A. aquasalis were used in this assay, in which their digestive tubes were removed submitted or kept down in solutions and centrifuged to obtain serum. Right away, they were examined with antisera of birds, mammals, reptiles and amphibians (lorosa et al. 1998

Statistical analyzes
The average of adult mosquitoes collected from January in 2006 to December in 2011, for both environments, were interpreted considering combinations with the average temperature indexes (°C), relative humidity (%) and precipitation (mm). In order to quantify the associations among the climate conditions and the presence of the species and the coexistence of species, we obtained the simple and partial correlation estimates using the software R (r DeveloPment Core team 2013).

Mosquito sampling
In total, 3,110 anopheline specimens were collected from adults and larvae distributed in nine species. The immature samples in the breeding sites totalized 190 anopheline larvae and five species collected. In Buriticupu, two breeding sites were positive and in São José de Ribamar, only one, out of a total of three, in each inspected locality.
The most frequent species was A. darlingi (41.1%) and was found only in Buriticupu, where it represented 78% of the sample. In São José de Ribamar, the most frequent was A. aquasalis (61.3%). The total of ILHH was 2.00, being 1.33 in Buriticupu and 0,64 in São José de Ribamar (Table 1).

Breeding site characterization
Regarding the water temperature of the breeding sites, very close values were found between, Buriticupu and São José de Ribamar, respectively 21ºC and 20ºC. The pH varied slightly between the environments, respectively 5.18 and 5.91. The electrical conductivity was 0.263 S/m1 for Buriticupu and 1.24 S/m-1 for São José de Ribamar. The total dissolved solids -TDS 131.00 mg/L in Buriticupu and 23.50 mg/L in São José de Ribamar. The salinity was lower than 0.0001 in Buriticupu and 0.5/1.000 in São José do Ribamar. In the chemical analysis of nitrite in Buriticupu, were found mean values 0.25 mg/L, while in São José de Ribamar, the mean values were 0.75. Regarding nitrate, the Buriticupu samples quantified 1.42 mg/L, while in São José de Ribamar, the values found were higher, 16.15 mg/L. Regarding the ammonia content in the breeding sites, the average value in Buriticupu was 4.63 mg/L. For São José do Ribamar, this value was 21.61 mg/L. The phosphorus content dissolved in Buriticupu breeding sites was on average 57.43 mg/L, and 55.03mg/L in São José do Ribamar (Table 3).
In relation to the presence of associated vegetation, the breeding sites in Buriticupu presented six families and six genera with greater dominance of the genus Cyperus sp. In São José de Ribamar, were found five families and five genera with Avicennia sp. and Rizophora sp. Just the genus Nymphaea was present in the breeding sites of both localities. All the evaluated breeding sites were classified as natural, in the soil, permanent or semi-permanent, whose margins are shaded due to the presence of Mauritia flexuosa, in Buriticupu and Avicennia sp. with Rizophora sp. in São José do Ribamar (Table 3).

Blood-feeding preferences
In Buriticupu, a total of 349 females were collected from A. darlingi engorged, 64 intradomicile, 139 in peridomicile, and 146 in extradomicile. About A. aquasalis 17 specimens were collected, 4 intradomicile and 13 in peridomicile. In intradomicile, 7.73% of A. darlingi collected, were fed human blood, followed by 5.17% with blood from birds and 2.86% with blood from cattle. In peridomicile, the highest number of A. darlingi fed with human blood (14.32), followed by dog blood (10.02%) and the third highest value for bovine blood (6.59%). The extradomicile data differ from the pattern observed in both environments, with the highest value for bird blood (13.75%), followed by human blood (12.32%) and cattle blood the third most frequent (6.59%). Considering the general total, the A. darlingi by human blood represented 34.38% of the samples, followed by bird -23.49% and cattle the third most frequent, representing 16.04% of the total. Whereas A. aquasalis, the percentage of specimens was very low, of which 13 specimens were found in peridomicile, fed mostly with human blood (58.82%) and another 4 in intradomicile fed with human blood (11.76%) and bird (11.76%) ( Table 4).
The analysis of the precipitin double reaction for bloodfeeding of A. darlingi and A. aquasalis allowed detecting that in intradomicile, 21 specimens (14.38%) of A. darlingi were fed on rodent / human blood and 17 (11.64%) on bird / human blood. Only one female from A. aquasalis fed on bird / human. In peridomicile, 19 specimens of A. darlingi (13.01%) fed on bird / human blood, while 14 (9.58%) fed on cat / bird and 6 (4.10%) on poultry / cattle blood. In extradomicile, 20 specimens from A. darlingi (13.69%) were engorged with human / skunk blood. There were also 18 specimens (12.32%) of the skunk / bird combination; 16 (10.95%) bird / cattle and 15 (10.27%) of the bird / dog combination. Considering the general total of the double reactions, the A. darlingi per bird / human, represented 24.65% of the mosquitoes collected, followed by the skunk / bird combination (21.91%) and 15.06% for the bird / cattle combination. For A. aquasalis reacted doubly only one specimen for the bird / human (Table 4).
The tests also revealed that five (20%) females caught in peridomicile had fed on birds. Considering the results of the double precipitin reaction, only three (20.00%) of the female caught in peridomicile revealed the combination of cat / human feeding and 12 specimens (80.00%) also in peridomicile the human / bird combination (Table 4).

Correlation between Anopheles species
In Buriticupu, we detected that A. darlingi coexists positively in significance with A. albitarsis s.l., A. oswaldoi, A. nuneztovari and A. evansae in Buriticupu. However, A. albitarsis s.l. was dominant on the same species, with the exception of A. darlingi. The behavior of these species reveals the potential of A. darlingi as the main vector in the environment and A. albitarsis s.l. as an associated vector. In São José de Ribamar, the coexistence of the species was significant for A. aquasalis and A. albitarsis s.l., and between A. oswaldoi and A. nuneztovari. The species A. darlingi and A. albitarsis s.l. coexist in that locality in a positive way among the evaluated environments, in the same way as A. oswaldoi and A. nuneztovari (Table 5).

Correlation between Anopheles species and climate variables
Of all the climatic data analyzed, only rainfall showed a significant correlation with A. darlingi in Buriticupu and with A. albitarsis s.l. and A. aquasalis in São José de Ribamar (Table   6). During the period of this study, in both localities, the monthly variations of temperature and humidity had few differences in amplitudes. However, the rainfall regime was decisive for the maintenance of A. darlingi in Buriticupu (P = 0.0280) ( Figure 1) and for A. aquasalis (P= 0.0051) (Figure 2) in São José de Ribamar. These data indicate that the presence of these species was strongly related to the rainy seasons in both localities.

Peak of hematophagy
The biting activity of the females in Buriticupu, behaved in a very similar way for the species A. darlingi, A. albitarsis s.l., A. nuneztovari and A. oswaldoi whose peaks occurred in the first two schedules in practically all of the evaluated environments.
Anopheles darlingi reached 7.0 mosquito/man/hour index in intradomicile between 6:00 and 7:00 pm and 10.9 between 7:00 and 8:00 pm. In peridomicile at the same time, the rates of mosquito/man/hour were 14.4 and 42.1, respectively. Already in extradomicile, were 6.9 and 10.9 mosquito/man/ hour. For A. albitarsis s.l. the highest indexes were reached in peridomicile between 6:00 and 7:00 pm and from 7:00 to 8:00 pm respectively, 7.0 and 10.0 mosquito/man/hour. In the species A. nuneztovari and A. oswaldoi the indexes were lower than one (Figure 3). In São José de Ribamar, only mosquitoes were collected during 12-hour samplings in a peridomicile environment. The mosquito/man/hour indexes were considered very low, varying between 0,00 and 0,07, in this case it wasn't possible to detect a well-defined period of the species' peak ( Figure 4).  Natural infection by Plasmodium spp.
The rates of natural infection could only be analyzed in Buriticupu, due to the more expressive volume of specimens captured. Only one pool was positive Plasmodium vivax and four for Plasmodium falciparum in Anopheles darlingi, whose MIR were respectively 0,6% and 2.2%. Anopheles albitarsis s.l. positivized a pool for P. vivax and MIR of 1.1%. Anopheles nuneztovari positivized for P. vivax and one for P. falciparum, whose MIR were 8.3% and 1.1 %. Anopheles oswaldoi was positive for P. falciparum and MIR 2.3% and also presented positivity for mixed infection, MIR 2.3% (Table 7). The analysis of the distribution of Anopheles spp. naturally infected by Plasmodium spp. guide us to the conclusion that A. darlingi was found infected in the three environments, ranging in the first four hours of the evening and closer to dawn. Anopheles albitarsis s.l. infected occurred in peridomicile and in extradomicile during the first two hours of the evening, respectively. Anopheles oswaldoi was found infected in intradomicile and peridomicile also, during the first two nocturnal times. Anopheles nuneztovari was found infected in the peridomicile between 10:00 and 11:00 pm ( Figure 5).   (2008) and lower than the controls of Ferreira et al. (2015) in artificial breeding sites, such as potholes and pisciculture tanks, respectively.

Study of behavioral patterns and infection analyses in anopheline…
The results enabled to verify that the anophelines fauna among home environments was more frequent in peridomicile. In this environment are located people at dusk and in the early hours of the night, as well as domestic animals and some occasional wild ones. In this context, it becomes possible to know the anophelines fauna, allowing to assess the level of risk of malaria transmission. Considering the densities of the vectors, the data found in Buriticupu, were more expressive than those obtained in the following municipalities: Paço do Lumiar (199 specimens), São Luiz ( moutinHo et al. 2011;BarBosa et al. 2014) and the island of São Luiz (Xavier & reBêlo 1999). In the rainy season, the availability of natural breeding sites increases, providing new breeding sites for anophelines, a hegemonic factor for expanding the densities of these mosquitoes.
Anopheles darlingi was the species that presented the greatest endophilic behavior, revealing the ability that this mosquito has to make the blood-feeding or hematophagy on humans   inside the houses. In addition, it was also the most frequent in peridomicile and extradomicile. The first scottophase schedules were the ones with the highest mosquito/man/ hour indexes with a strong trend of bimodal behavior in Buriticupu. These behaviors were similar to those observed in works carried out in other locations in the Amazon (Deane 1986;lourenço-De-oliveira et al. 1989;taDei et al. 1993;taDei et al. 2000;taDei et al. 2017). Nevertheless, the biting activity of A. aquasalis during nighttime intervals was also reported in peridomicile (Xavier & reBêlo 1999). In general, mosquito/ man/hour indexes of the vectors differ greatly in each region, at certain times and between species (lourenço- De-oliveira et al. 1989;GalarDo et al. 2009;Póvoa et al. 2009;BarBosa et al. 2016). Whereas the peridomicile was the most affluent and abundant species occurrence environment, allied to human habits in the main peak times of mosquitoes, this information points to guidance of differentiated control measures in this mean, since it has an important role in malaria transmission (vezeneGHo et al. 2016).
It should be noted that in São José de Ribamar, five specimens of A. darlingi were found. The record of A. darlingi in the island of São Luís had been made by Deane et al. (1948) in which they stated: "In an experiment done in 1941, freshly hatched larvae of A. darlingi captured in São Luiz (Maranhão)" […]. The data of this work corroborate this affirmation because since that publication, no record of the species had been done in that island. Another important fact was the first record of A. aquasalis in Buriticupu; 300 km distant from the Atlantic coast (Barros et al. 2015).  The blood feeding of the two main vectors, A. darlingi and A. aquasalis allow to infer that they are very eclectic species feeding on several hosts. However, the higher frequencies of the reactions revealed that both presented a remarkable anthropophilic behavior, as observed in the experiments conducted by Deane et al. (1949). In São José de Ribamar, samplings proved the preference of A. aquasalis to feed on humans (80%). At this location the other animals hosts were birds and cats. This data differs from what was found by Flores-menDoza et al. (1996), in Rio de Janeiro, who emphasized that this species exhibits zoophilic behavior. However, Deane (1986), points out that the vector capacity of this species is related to the density of the population, whose effects are more pronounced in the north and northeast coast of Brazil.

Study of behavioral patterns and infection analyses in anopheline…
The natural infection detected four species infected with Plasmodium ssp. in Buriticupu -A. darlingi, A. albitarsis s.l., A. oswaldoi and A. nuneztovari. Notoriously, A. darlingi, stands out being infected with both P. vivax and P. falciparum in the three environments analyzed, reason why it is considered the Volume 13, 2020 -www.periodico.ebras.bio.br EntomoBrasilis 13: e0820  In the present study, the three main vectors of malaria in Brazil were detected in the evaluated areas: A. darlingi, A. aquasalis and A. albitarsis s.l., whose densities justify their dominance in the environment. Secondary vectors, such as, A. nuneztovari and A. oswaldoi were also found, however, in a smallest proportion. Nevertheless, as a whole, except for e-ISSN 1983-0572 A. aquasalis, all of them had natural infections by Plasmodium ssp. Therefore, have a relevant role in the transmission of malaria, in the municipalities of Maranhão. The volume of information collected allows us to state that Anopheles darlingi remains as the main vector of malaria in Buriticupu. As A. aquasalis probably has important highlight in the transmission of the disease in São José de Ribamar.

Study of behavioral patterns and infection analyses in anopheline…
Thus, both locations a high risk for malaria transmission. Finally, the data collected by this work contribute to elucidate a part of the transmission chain in both localities, as well as, serve to guide the control actions by the public health service and the population in general, so that they can adopt conduits to prevent new infections.
In addition, these data are important because they also contribute to the process of controlling and eliminating malaria, an activity which is relevant today, in the Amazon region. The areas of occurrence records of the species of Anopheles are the references for the implementation of specific actions, whose ultimate goal is the elimination of this disease, initially focusing on the P. falciparum (santelli et al. 2016).